Programmed cell death or apoptosis is a physiological process by which genetically damaged cells or undesired cells can be eliminated. Various morphological and molecular changes undergoing during the process of apoptosis are the formation of apoptotic blebs of the cell membrane, cell shrinkage, condensation of chromatin and the disruption of deoxyribonucleic acid (DNA) into typical fragments of multiples of 180 base pairs. These changes can be detected in a number of ways. DNA ladder formation, which is observed following gel electrophoresis technique although is widely accepted but does not reflect the DNA breakdown in individual cell and also may miss contributions from small sub-populations in a heterogeneous cell population. Alkaline comet assay as measured by single cell gel electrophoresis, on the other hand, accurately measures DNA fragmentation on a single cell level and allows analysis of subpopulation of cells. The assay was originally developed for measuring DNA damage of cells exposed to any genotoxic agent. However, the comet image generated by an apoptotic cell is different from that obtained with a cell treated for a short time with a genotoxic agent. Correlation of comet formation with various other established parameters of apoptosis is very important. The present study aims to correlate different features of apoptosis with the formation of comet tail in human leukemia K-562 cells using tea extracts. Apoptosis as measured by formation of apoptotic bodies, flow cytometric analysis, activation of caspase 3 and 8, and expressions of apoptosis related genes such as bcl-2 and bax showed high degree of correlation with comet tail moment. This indicates that comet assay can accurately reflect measure of DNA fragmentation and hence can be used to detect a cell undergoing apoptosis.

译文

程序性细胞死亡或凋亡是一种生理过程,通过该过程可以消除遗传受损的细胞或不希望的细胞。在细胞凋亡过程中发生的各种形态和分子变化是细胞膜凋亡泡的形成,细胞收缩,染色质的浓缩以及脱氧核糖核酸 (DNA) 破坏成多个180碱基对的典型片段。可以通过多种方式检测这些变化。通过凝胶电泳技术观察到的DNA阶梯形成,尽管已被广泛接受,但并未反映单个细胞中的DNA分解,并且可能会错过异质细胞群中小亚群的贡献。另一方面,通过单细胞凝胶电泳测量的碱性彗星分析可以在单细胞水平上准确地测量DNA片段化,并可以分析细胞亚群。该测定法最初是为测量暴露于任何遗传毒性剂的细胞的DNA损伤而开发的。然而,由凋亡细胞产生的彗星图像与用遗传毒性剂处理短时间的细胞获得的图像不同。彗星形成与其他各种已建立的凋亡参数的相关性非常重要。本研究旨在使用tea提取物将细胞凋亡的不同特征与人类白血病K-562细胞中彗尾的形成相关联。通过凋亡小体的形成,流式细胞术分析,caspase 3和8的激活以及凋亡相关基因 (例如bcl-2和bax) 的表达来测量凋亡,与彗星尾矩高度相关。这表明彗星分析可以准确反映DNA片段的测量,因此可以用于检测细胞凋亡。

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